PROJECT SUMMARY Zinc is an essential nutrient because it is a required cofactor for many proteins. Therefore, cells have evolved with efficient mechanisms to maintain zinc homeostasis during zinc deficiency. Cells can also alter specific metabolic processes to adapt to zinc deficiency. Among eukaryotes, these mechanisms are best understood in the yeast Saccharomyces cerevisiae. In this yeast, the Zap1 transcription factor is the central regulator of zinc homeostasis. Over the years, analysis of Zap1 and its target genes has led to many key discoveries about how cells survive and thrive during zinc deficiency. In this application, three specific aims are proposed that build on that solid foundation of prior work. Translation of the mRNAs for zinc-binding proteins generates apoproteins that rapidly bind their metal cofactor to become stably folded. During zinc deficiency, we propose that zinc proteins are synthesized but are largely unable to bind the metal because of its limited supply. The resulting accumulation of unmetalated apoproteins greatly disrupts protein homeostasis. We have discovered many mechanisms cells use to diminish and adapt to this stress. In Aim 1, we will further test the hypothesis of abundant apoproteins by identifying specific zinc proteins that are not metalated during zinc deficiency. These studies will provide unprecedented insights into the trafficking of zinc within cells. We will then determine the role of the Tsa1 protein chaperone in stabilizing those apoproteins so that they can be efficiently metalated when zinc levels increase. Tsa1 is critical for growth during zinc deficiency and these studies will define the molecular basis of this protein?s important function. In Aim 2, we will investigate the role of the proteasome- ubiquitin system in degrading apoproteins and the role of protein quality control compartments (IPOD, JUNQ) in sequestering misfolded apoproteins and mediating their degradation by autophagy. These studies will establish an integrated model of protein homeostasis during zinc deficiency. Finally, in Aim 3, we will test how the model of protein homeostasis during zinc deficiency that we have generated for yeast applies to human cells. We will assess whether the large abundance of apoproteins during zinc deficiency is an evolutionarily conserved stress and we will test the role of human Tsa1 orthologs in tolerating that stress and facilitating the binding of zinc when its supplies increase. This research has clear relevance for human health because zinc deficiency is a common nutrient deficiency in the US population. Our analysis of the effect of zinc deficiency on protein homeostasis may ultimately lead to fundamental insights into diseases of protein misfolding such as amyotrophic lateral sclerosis (ALS), Parkinson?s, Alzheimer?s, and prion diseases and their relationships with metal homeostasis. In addition, zinc homeostasis in fungi and other microbes is critical for pathogenesis. We are illuminating processes that may be targeted by anti-fungal therapies. !